I am measuring levels on mirco RNAs in a murine myoblast cell line. So, I collected the cells and processed them for small RNA extraction using MirVana Kit. Upon estimating the concentration and purity (260/280) using the nanodrop machine, I received a negative concentration value for most samples with poor purity.
I was supposed to go ahead with making cDNA and running it through qPCR to analyse the levels. I have two questions -
1. What could have gone wrong? This is the first time I am receiving such values.
2. Is it worth going ahead with cDNA and qPCR?
Prabhu, the kit comes with a total RNA extraction and further gives the steps for small RNA extraction. On my previous attempts, I used to perform total RNA extraction followed by small RNA and the results were fine. I used to get a small RNA concentration of ~ 30 ng/uL. But, this time it went awry. Just so you know, I have kept everything else - the kit, the reagents, my technique - steady.
Prabhu, thanks for following up. I am using TaqMan MicroRNA Assays kit by applied biosystems. The recommendations state that the kit is sensitive enough and requires between 1 and 10 ng of total RNA or equivalent. What do you make of it?
Prabhu, so that would mean stopping at the total RNA extraction step and proceeding with the small RNA purification protocol. Fair enough. I will give it a go.
Saurabh,
As you are using a spectrophotometer to measure your sample, you'd better use 20 ng of RNA to prepare your cDNA, because the spectrophotometer for such small quantities is not the best method of quantitation.
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